The present invention is in the field of protein crystallization and in particular protein crystallization of interferons.
The human interferon alphas are a family of proteins comprising at least 24 subspecies, Zoon K. C., Interferon 9:1 (1987), Gresser I., ed. Academic Press, N.Y. They were originally described as agents capable of inducing an antiviral state in cells but are known as pleiotropic lymphokines affecting many functions of the immune system, Opdenakker, et al., Experimentia 45:513 (1989). Apart from their in vitro biological activities the human interferon alphas are currently used for several indications, e.g., hairy cell leukemia, Kaposi's Sarcoma, venereal warts, hepatitis B and hepatitis C.
Interferon alpha-2b is prepared as a purified sterile, lyophilized recombinant interferon formulation. An example of a commercially available interferon alpha-2b is INTRON A.RTM. produced by Schering-Plough Corporation, Kenilworth, N.J. The demand for highly purified and crystalline forms of interferon alpha, especially the recombinant type alpha-2b, is of foremost importance for structure elucidation as well as for formulation of various dosage forms including the development of controlled release formulations.
Two forms of crystalline human interferon alpha have been reported, namely from Miller et al., Science, 215:689 (1982); Kung et al., U.S. Pat. No. 4,672,108; Weissmann, The Cloning of Interferon and other Mistakes, In: Interferon 1981, Ian Gresser, ed., Academic Press, N.Y., 101-134 ; Weissmann, Phil. Trans. R. Soc. Lond. B299:7 (1982); Nagabhushan, et al., `Characterization of Genetically Engineered alpha-2 Interferon`, In: Interferon: Research Clinical Application and Regulatory Consideration, Zoon et al., Elsevier, N.Y. 79 (1982). These publications describe methods for crystallizing interferon alpha-2 in polyethylene glycol at low temperature or in a phosphate buffer solution by adjusting the pH or temperature. The Miller et al. article also mentions crystalline alpha-2 in a "prismatic form". Conditions for producing monoclinic prismatic crystals of interferon alpha-2b from solutions of ammonium sulfate in vapor diffusion hanging drop experiments at 22.degree. C. are disclosed in International Patent Application No. PCT/US 91/03660.
IFN-alpha is generally administered either by subcutaneous or intravenous injection usually in hospital or clinical settings. IFN-alpha has a serum half-life of 2-6 hours when injected subcutaneously or minutes when injected intravenously, and characteristically shows a "burst" or a "pulse" (i.e., a rapid blood serum level rise followed by a rapid blood serum level clearance) profile when blood levels are measured over time. Thus frequent administration of doses of the protein must be made to maintain a therapeutically effective blood serum concentration of the drug. There are clinical situations when it may be therapeutically more advantageous to develop an IFN-alpha formulation in which the protein is continuously released into the blood stream so that the serum concentration of the protein reaches a plateau and remains at that level for a period of time. This is an example controlled release formulation.
To date none of the known crystalline IFN-alphas have shown properties desirable for a controlled drug delivery system, in particular, limited solubility at 37.degree. C. and stability in a `Generally Recognized as Safe` (GRAS) category formulation suitable for injection. There are a number of potential advantages of a controlled release therapeutic. Primarily, controlled release drugs can be administered at lower effective doses which improves their safety while maintaining or improving their efficacy. New therapeutic indications can be explored because prolonged bioavailability offers the opportunity for increased biodistribution to enhance tissue and organ penetration.
There is thus a need for a controlled-release formulation of IFN-alpha.